Speaker
Description
Introduction
Three-dimensional (3D) cell culture techniques have become particularly important for the study of lymphoma cells, which are non-adherent and difficult to culture in traditional two-dimensional (2D) systems, and provide cellular environments that better mimic the in vivo tumour microenvironment. Conventional 2D cultures fail to replicate critical features such as cell-cell and cell-matrix interactions and chemical gradients, leading to discrepancies in drug sensitivity assessments. Co-culturing non-adherent lymphoma cells with adherent fibroblasts presents additional challenges due to intrinsic differences in growth behaviour, adhesion properties and microenvironmental requirements. Nevertheless, the establishment of such a co-culture system is crucial to mimic the complexity of the tumour microenvironment. To address these challenges, we developed a 3D culture system using hemispherical hydrogel structures, termed 'cell domes', immobilised on glass plates.
Materials and Methods
Cell Domes were fabricated by first forming a hemispherical gelatin hydrogel containing diffuse large B-cell lymphoma (DLBCL)-derived KML-1 cells and human dermal fibroblasts (HDFs) on phenol-modified glass plates. A hydrogel membrane composed of alginate and gelatin derivatives was then formed on the hemispherical hydrogel by horseradish peroxidase (HRP)-mediated crosslinking. The internal cavity structure containing the two cells was achieved by incubating the constructs at 37°C, inducing a gel-to-sol transition of the gelatin core. Cell proliferation, hypoxic status, surface marker expression (CD20) and drug sensitivity to doxorubicin were evaluated.
Results and Discussion
KML-1 and HDF cells, initially dispersed within the cell domes, gradually formed aggregates over 10 days, as confirmed by microscopy and section analysis. A hypoxic gradient developed within the domes, with increased HIF-1α expression observed towards the centre of the aggregates, replicating a key feature of in vivo lymphoma tissue. Flow cytometry revealed a decrease in CD20 expression in 3D cultured KML-1 cells compared to 2D cultures. In addition, drug sensitivity assays showed that 3D co-cultured cells were more resistant to doxorubicin, highlighting the influence of the 3D microenvironment on therapeutic response.
Conclusion
The Cell Dome system enables effective 3D co-culture of non-adherent lymphoma cells and adherent fibroblasts, overcoming the traditional challenges associated with their different biological properties. This approach successfully recapitulates critical features of the tumour microenvironment such as hypoxia and drug resistance, providing a promising platform for physiologically relevant preclinical lymphoma research and therapeutic evaluation.
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